CN105754928B - A kind of separation of prawn embryonic cell and cultural method - Google Patents
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Abstract
The present invention establishes a kind of effective prawn embryonic cell dissociation and culture technique, and being applied to prawn embryonic cell to build is research.Present invention optimizes the Aseptic sterilisation processing techniques of prawn embryo, shrimp ovum directly can be collected from prawn seed-rearing factory, transport laboratory post-processing, the especially optimization of the concentration for the treatment of of Iodophor back, not only guaranteed effectively to control microorganism and protozoic pollution, but also utmostly reduced Iodophor to the toxicity of embryo；The formula of prawn complete medium is optimized, provides more suitable nutritional condition for the adherent and growth of prawn embryonic cell, adherent faster growth is more preferable, while simplifying the formula of culture medium, reduces the cost of culture medium；Using the embryonic cell collector made by appropriate aperture stainless steel filtering net, the method for smashing prawn embryo to pieces can obtain embryonic cell and cell mass largely living in a short time, it is easy to operate quick, the several seconds is only used, not easy to pollute, the remote super reported other dissociating methods of effect.
Description
Technical field
The invention belongs to zooblasts, technical field of tissue culture, and in particular to sterile point of a kind of prawn embryonic cell
From with extracorporeal culturing method.
Background technique
The frequent outburst of prawn virus disease brings heavy blow to shrimp culture industry, and becoming restriction shrimp culture industry can
The bottleneck problem of sustainable development.Establishing for prawn immortalized cell line can be isolating and purifying, studying its machine that causes a disease for prawn's virus
It manages, the effective research means of the offers such as efficient antiviral vaccine or drug and carrier is provided.Although domestic and international experts and scholars exist
Prawn cell, which is built to fasten, has carried out a large amount of exploration and effort, but due to the cultured cell in vitro in prawn adult tissue source regardless of
It splits, it is difficult to be converted, immortalized cell line fails always foundation.And body early embryo due to active proliferation ability and
More differentiation potentials are that prawn cell builds the good organization source for being.But prawn fertilized eggs are isolecithal egg, holoblastic cleavage, morning
The separation of phase embryonic cell and culture are difficult, and easily pollute, thus in relation to prawn embryonic cell culture in terms of research report
Seldom.Most earlier than 1996, the bright grade of virgin skirt, Toullec etc. and Frerichs report Chinese prawn, Indian prawn and sieve respectively
The embryonic cell cultivation results of family name pond crayfish.The bright equal trial in Chinese prawn embryonic cell culture of virgin skirt is because of microorganism and primary
Animal it is serious pollution and fail.Toullec etc. using Indian prawn 16- cell stage embryo as material, using M199 culture medium and
Drug-treated removes the dissociating method of fertilization membrane, successfully obtains the originally culture embryonic cell of adherent growth, but cell monolayer is only
It maintains 2 weeks.Frerichs is using 7-13 days embryos of Macrobrachium rosenbergii as material, using M199 culture medium and polishing, also successfully
To the originally culture embryonic cell of adherent growth, but not formed continuity single layer, and after Mechanical Method passage, cell no longer pastes
Wall.After 6 years, Fan Tingjun etc. (2002) is using the MPS culture medium and polishing for being added to growth factor (bFGF and IGF-II), training
It supports and has obtained Chinese prawn embryonic cell continuity single layer, but continue the report for being tied to form function after having no, result is not also by him
People repeats to come out.After 7 years, remaining dawn etc. (2009) explores dissociation and the culture side of Crustin blastaea and primitive gut blastocyte
Method, discovery post-mortem method are suitable for separation blastomere, and blood counting chamber pressed disc method is suitable for separation primitive gut blastocyte, the embryo cultivated
Fetus cells single layer can maintain in vitro 1-5 weeks, but have no had significant proliferation.Post-mortem method and the pressed disc method efficiency that operates are very low, easily
Pollution, and polishing, crossing sieve method and the skill of handling needles excessively cannot obtain enough living cells.As it can be seen that realize prawn embryo from now on
The longterm culture in vitro of cell so that build and be tied to form function, need to establish dissociation and the cultural method of effective prawn embryonic cell with
And aseptic technique.
Summary of the invention
The purpose of the present invention is establishing a kind of effective prawn embryonic cell dissociation and culture technique, and it is applied to pair
It is research that shrimp embryonic cell, which is built,.
Present invention firstly provides a kind of cleaning methods of prawn body early embryo, the specific steps are as follows: collects prawn with suction pipe
Into centrifuge tube, antiseptic sea water washs 5-6 times embryo, reject dead embryo and impurity particle；Embryo transfer is stainless to 70 mesh
In steel strainer, eluriate for several times after, flowing water continuous flushing 1-2h, then with 10% Iodophor handle 5-10 minute, removal bacterium with very
The pollution of the microorganisms such as bacterium；75% alcohol of short duration rinsing 20-30 seconds removes Iodophor；Then successively with green added with 2000IU/mL
The antiseptic sea water and PBS of mycin and streptomysin sufficiently rinse, and remove alcohol；It is finally temporarily stored into prawn basal medium.
The preparation method of above-mentioned antiseptic sea water: natural sea-water successively after 16-20 layers of gauze and 0.22 μm of membrane filtration,
High pressure steam sterilization 20 minutes.
The preparation method of above-mentioned PBS solution: 8.0g NaCl, 0.2g KCl, 3.0g Na are weighed respectively2HPO4·12H2O and
The KH of 0.2g2PO4Be dissolved in 1L pure water, dissolve, with 3M NaOH adjust pH value to 7.2~7.4,121 DEG C high pressure sterilization 20 minutes,
It is saved backup for 4 DEG C after packing.
The preparation method of above-mentioned prawn basal medium: 20.55g L-15 culture medium, 5g are dissolved in 1 liter of ultrapure water
NaCl, 2g glucose, 1g NaHCO3, 0.01g taurine, 0.01g proline, 1.0 × 105IU Benzylpenicillin sodium salt and 100mg sulfuric acid
Streptomysin adjusts pH value to 7.2-7.4, with 0.22 μm of filtering with microporous membrane degerming, packing, -20 DEG C of preservations.
The invention further relates to a kind of methods that stainless steel filtering net smashs method separation prawn embryonic cell to pieces.Specific steps: it will protect
In the prawn embryo transfer to sterilized mortar deposited, the extra culture medium of reject；With sterilized embryonic cell collector (patent
Number: 201420430124.9) curved stainless steel strainer squeezes prawn embryo to smash fertilization membrane to pieces, keeps embryonic cell free out
Come；Fresh prawn basal medium is taken, stainless steel filtering net is rinsed, collects all embryonic cells and cell mass, with prawn basis
Culture medium, sedimentation is washed 1-2 times, until clear, finally uses prawn complete medium suspension embryonic cell and cell mass, connect
Kind is into culture bottle or culture plate.
The preparation method of above-mentioned prawn complete medium: often going up and state in prawn basal medium, adds 8% tire ox blood
Clearly, 20 μ g basic fibroblast growth factors (bFGF) and 20 μ g epidermal growth factor (EGF), pH7.2-7.4.With 0.22 μm
Filtering with microporous membrane degerming, packing, -20 DEG C of preservations.
The invention further relates to a kind of cultural method of prawn embryonic cell, specific steps: prawn embryonic cell suspension is connect
Kind into culture bottle or culture plate, in 28 DEG C, 3%CO2Stationary culture in incubator.Observation cell adherent growth situation daily,
According to culture medium color change, every 2 days 1/2 culture mediums of replacement.
The invention further relates to a kind of propagating method of prawn embryonic cell, specific steps: washing cell monolayer with 4 DEG C of PBS,
Then it is digested with the digestive ferment HyQTase of nonmammalian source to cell and takes off wall, prawn complete medium is then added and is resuspended
Cell, centrifuge washing 1 time, inoculation passage.
The present invention has the advantages that optimize and establish the Aseptic sterilisation processing technique of prawn embryo, it can be directly from prawn
Shrimp ovum is collected by nursery factory, transports laboratory post-processing back, and the especially optimization of the concentration for the treatment of of Iodophor had both guaranteed effectively to control micro- life
Object and protozoic pollution, and Iodophor is utmostly reduced to the toxicity of embryo, realize 100% sterile success rate；It optimizes
The formula of prawn complete medium provides more suitable nutritional condition for the adherent and growth of prawn embryonic cell, it is adherent more
Fastly, growth is more preferable, while simplifying the formula of culture medium, reduces the cost of culture medium；It is filtered using by appropriate bore diameter stainless steel
Net the embryonic cell collector of production, the method for smashing prawn embryo to pieces, can obtain in a short time largely living embryonic cell and
Cell mass, easy to operate quickly only to use the several seconds, not easy to pollute, the remote super reported other dissociating methods of effect.
Detailed description of the invention
Fig. 1: prawn embryo's flusher schematic diagram.
Fig. 2: four kinds of prawn embryonic cell separation method schematic diagrames.A: stainless steel filtering net smashs method to pieces；B: cell sieve filtration method；
C: grinding rod smashs method to pieces；D: homogenate method.1: mortar；2:60-70 mesh stainless steel filtering net；3:200 mesh Nylon cell sieve；4:50mL from
Heart pipe；5:20mL syringe cork；6:1.5mL centrifuge tube；7: grinding rod；8: tissue grinder.
The cultivation results of 0-48h after tetra- kinds of physical method dissociation Marsupenaeus japonicus body early embryo cells of Fig. 3.A: prawn appendage bud
Phase embryo；B and B1: homogenate method；C, C1 and C2: grinding rod smashs method to pieces；D, D1 and D2:200 mesh screen method；E, E1 and E2: no
Rust steel strainer smashs method to pieces.
The cultivation results of 3-48h after the isolated Marsupenaeus japonicus embryonic cell inoculation of Fig. 4 stainless steel filtering net crush method.
A: originally culture 3h, B: originally culture is for 24 hours；C: originally culture 36h；D: originally culture 48h.
Cultivation results of Fig. 5 Marsupenaeus japonicus embryonic cell in control medium.It is added in the control medium
10% prawn Muscle vector and 15% fetal calf serum.A: originally culture 3h, B: originally culture is for 24 hours.
Fig. 6 cell injuring model 3-20 days results of Marsupenaeus japonicus body early embryo.A: originally culture 3d, B: originally culture
5d；C: originally culture 7d；D: originally culture 10d；E: originally culture 15d；F: originally culture 20d.
The differentiation of Fig. 7 prawn originally culture embryonic cell.A: the differentiation of fibroblast-like cells (F)；B: epithelioid cell (E)
Differentiation；C: the differentiation of neural-like cells (N)；D: the differentiation of cardiac muscle cell (M).
The secondary culture result of Fig. 8 Marsupenaeus japonicus embryonic cell.A: originally culture embryonic cell before passing on；B:4 DEG C of PBS
With the embryonic cell of adherent growth after the passage of HyQTase digestion method.
Specific embodiment
Preferably to explain the present invention, by taking the separation of the embryonic cell of Marsupenaeus japonicus is with culture as an example, this is further illustrated
The main contents of invention:
The cleaning and disinfection treatment technology of 1 Marsupenaeus japonicus body early embryo of embodiment.
Specific step is as follows:
(1) preparation of sterilizing seawater: fresh natural sea-water successively passes through 16-20 layers of gauze and 0.22 μm of membrane filtration
Afterwards, high pressure steam sterilization 20 minutes, it is spare after cooling.
(2) the appendage bud phase embryo of 6-8h after oviposition, ocean temperature the materials of prawn body early embryo: are collected from prawn seed-rearing field
It 28-30 DEG C, is fitted into polybag, is oxygenated, tightens sack, be placed in bubble chamber, transport laboratory back.
(3) collection of prawn embryo: standing a few minutes, after shrimp ovum is deposited to bottom, with plastic suction pipe by prawn embryo
It is collected into 50mL centrifuge tube, sterilizes seawater flushing 5-6 times, during the rinsing process inhale the embryo for suspending dead and impurity particle
Out.
(4) washing of prawn embryo: being to carry out elutriation 3- to embryo in 70 mesh stainless steel filtering nets by embryo transfer to bottom
5 times, then flowing water repeated flushing 1-2h, removes the pollution of algae, zooplankter and protozoan etc., ensures in flushing process every
Piece embryo is resuspended in completely in sterilizing seawater.Embryo's flusher is as shown in Fig. 1.
(5) disinfection treatment of prawn embryo: flowing water flushing finishes, and in superclean bench, prawn embryo transfer is arrived
It is disinfected 5-10 minutes in 10% disinfectant tamed iodine, removes the pollution of the microorganisms such as bacterium and fungi, during which constantly use sterile
Suction pipe pressure-vaccum embryo repeatedly, to ensure that every piece of embryo is completely submerged in thimerosal.Embryo is then completely immersed in 75% alcohol
In of short duration rinsing 20-30 seconds, remove Iodophor, then successively use 2000IU/mL dual anti-(penicillin and streptomysin) antiseptic sea water
It is sufficiently rinsed with PBS, removes alcohol；It is finally temporarily stored into prawn basal medium.
The pollution of microorganism, algae, protozoan and zooplankter etc. can be effectively controlled for the method, it can be achieved that 100% is sterile
Success rate, and prawn embryo can be resistant to the processing of Iodophor and alcohol well, keep activity.
The preparation method of above-mentioned prawn basal medium: 20.55g L-15 culture medium, 5g are dissolved in 1 liter of ultrapure water
NaCl, 2g glucose, 1g NaHCO3, 0.01g taurine, 0.01g proline, 1.0 × 105IU Benzylpenicillin sodium salt and 100mg sulfuric acid
Streptomysin adjusts pH value to 7.2-7.4, with 0.22 μm of filtering with microporous membrane degerming, packing, -20 DEG C of preservations.
The preparation method of above-mentioned PBS solution: 8.0g NaCl, 0.2g KCl, 3.0g Na are weighed respectively2HPO4·12H2O and
The KH of 0.2g2PO4Be dissolved in 1L pure water, dissolve, with 3M NaOH adjust pH value to 7.2~7.4,121 DEG C high pressure sterilization 20 minutes,
It is saved backup for 4 DEG C after packing.
The screening of the best dissociating method of 2 Marsupenaeus japonicus embryonic cell of embodiment.
Embryo's outer layer of prawn has fertilization membrane to lift the chorion to be formed, to protect embryo from the injury of external environment.It wants
The body early embryo cell of acquisition dispersion is thought it is necessary to be crushed the tough and tensile chorion of its outer layer elasticity.Since prawn fertilized eggs are isolecithal egg,
Holoblastic cleavage, the separation and culture of body early embryo cell are difficult.The present invention compares the separation and training of 4 kinds of physics dissociating methods
Effect is supported, as illustrated in figs. 2-3, the separating effect that discovery stainless steel filtering net smashs method to pieces is best, and the complete of largely work can be obtained
Embryonic cell and cell mass.
(1) stainless steel filtering net smashs method to pieces: in the prawn embryo transfer to sterilized mortar that aseptic process is crossed, reject is more
Remaining culture medium；Holding sterilized embryonic cell collector, (patent No.: 201420430124.9), the front end of the tool is outside one
Convex stainless steel filtering net, mesh diameter are slightly smaller than shrimp ovum diameter, and vertical extruding prawn embryo is multiple, smash chorion to pieces, make embryo
Cell free comes out；Prawn basal medium is taken, the inside and outside wall of stainless steel filtering net is rinsed, collects all embryonic cells and cell
Group, with prawn basal medium, sedimentation is washed 1-2 times, until clear, finally thin with prawn complete medium suspension embryo
Born of the same parents and cell mass are seeded to stationary culture in culture bottle or culture plate.
(2) cell sieve filtration method: squeezing the embryo that is laid in 200 mesh cell sieves with asepsis injector cork, with complete
Culture medium rinses cell sieve, filters embryonic cell from sieve pore, collects the cell suspension inoculation of filtration to culture bottle or culture
Stationary culture in plate.
(3) grinding rod smashs method to pieces: squeezing the embryo of 1.5mL centrifugation bottom of the tube with the thick head of grinding rod, smashs to pieces to without complete
Embryo particle.After being resuspended with prawn complete medium, it is seeded to stationary culture in culture bottle or culture plate.
(4) it is homogenized method: the prawn embryo transfer that aseptic process is crossed to 1.5mL centrifuge tube bottom, organizes homogenizer 2000rpm
It is homogenized 3min, until stopping homogenate without complete embryo particle.Tissue homogenate is transferred in 15mL centrifuge tube, 1000rpm from
Heart 5min abandons supernatant, precipitating is resuspended in prawn complete medium, stationary culture in culture bottle or culture plate is seeded to.
The preparation method of above-mentioned prawn complete medium: often going up and state in prawn basal medium, adds 8% tire ox blood
Clearly, 20 μ g basic fibroblast growth factors (bFGF) and 20 μ g epidermal growth EGF, pH 7.2-7.4.With 0.22 μm of micropore
Membrane filtration degerming, packing, -20 DEG C of preservations.With patent: " a kind of Prawn cell culture medium " (patent No.:
ZL201310162351.8 it) compares, in the formula of prawn complete medium according to the present invention, does not add any prawn muscle
Extracting solution, the additive amount for reducing fetal calf serum is 8%, rather than 15%.
The dissociation result of embryonic cell is as shown in figure 3, only stainless steel filtering net smashs method to pieces and can get a large amount of individual cells
And cell mass, cell state is good, and adherent rate and high survival rate can form cell monolayer, and the service life is long, and can survive 23 days left sides in vitro
It is right.And cell sieve filtration method can get individual cells, but cannot obtain cell mass, isolated individual cells limited amount and
It is adherent less able, cell monolayer cannot be formed.Grinding rod smashs that method is separable to obtain individual cells and cell mass to pieces, but cellular
State is poor, cannot form cell monolayer.Tissue homogenate rule cannot obtain cell living.
The cultural method of 3 Marsupenaeus japonicus embryonic cell of embodiment.
Specific steps: prawn embryonic cell suspension is inoculated into culture bottle or culture plate, in 28 DEG C, 3%CO2Incubator
Middle stationary culture.Observation cell adherent growth situation daily, according to culture medium color change, every 2 days 1/2 culture mediums of replacement.
As shown in Fig. 4, stainless steel filtering net smashs the isolated embryonic cell of method and cell mass to pieces from after being inoculated with 2-3 hours
Can adherent growth, cell becomes elongated and drawout, hence it is evident that observes the presence of a large amount of yolk proteins, cells show is obvious out
Growth tendency.The iuntercellular of adherent growth contacts with each other in adjacent big cell mass after 48 hours, reaches half convergence state, this
When yolk protein amount significantly reduce.
Whether prawn complete medium provided by the present invention is to adding prawn Muscle vector and fetal calf serum adds
Dosage is optimized, and prawn Muscle vector is not added in discovery, while reducing the additive amount (8%) of fetal calf serum, more favorably
In the adherent and growth of prawn embryonic cell and cell mass.Control medium used in attached drawing 5 is patent: " a kind of prawn is thin
Prawn complete medium involved in born of the same parents' culture medium " (patent No.: ZL201310162351.8).From in attached drawing 4 as it can be seen that inoculation after
2-3h, embryonic cell and cell mass all adherent growths.And in attached drawing 5,2-3h after inoculation, only single embryonic cell patch
Wall, cell mass is not yet adherent, still suspends.And for 24 hours after, the quantity that cell is moved out in attached drawing 5 is also obviously made a farfetched comparison in Fig. 4
It is few.As it can be seen that prawn complete medium provided by the present invention is more conducive to the adherent of prawn embryonic cell and growth, simplify simultaneously
The formula of culture medium reduces the cost of culture medium.
As shown in Fig. 6, started the cell for occurring that there is rhythm and pace of moving things contractile function, and rhythmicity at cell culture the 3rd day
It shrinks sustainable one week or so, cell just maintains stationary state until Apoptosis later.When embryonic cell culture was to the 5th day
It can reach the convergence state of 70%-80%, but be proliferated unobvious.When cell culture was to the 20th day, the adherent ability of cell compared with
The cell of early stage culture significantly reduces, and vacuolation is deepened and presented to part cellular colours, and cell quantity significantly reduces, fragment of tissue
Increase with black pointing object, subsequent Apoptosis.
As shown in Fig. 7, Marsupenaeus japonicus body early embryo cell can be cultivated continuously 20 days or more in vitro, but the after being inoculated with
Cell differentiation phenomenon can be observed within 3 days.Mainly there are 3 seed type noble cells: fibroblast-like cells, epithelioid cell and neural sample
Cell.Wherein, part fibroblast-like cells after inoculation start have the function of Rythmic contractions characteristic within the 3rd day, this rhythmicity
It shrinks sustainable one week or so, cell is just restored to stationary state later, until Apoptosis.
The secondary culture technology of 4 Marsupenaeus japonicus embryonic cell of embodiment.
Specific steps:
(1) cell culture of Marsupenaeus japonicus body early embryo was to the 5th day, and when cell grows up to single layer, (cell confluency degree is 70%-
80%) when, to 25-cm24 DEG C of PBS of 1mL are added in Tissue Culture Flask and are incubated for 30sec or so suction.
(2) 1mL HyQTase digestive juice is added and digests 4min, microscopically observation is until 80% or so cellular contraction becomes
Bowlder terminates digestion.4mL Prawn cell culture medium is added into Tissue Culture Flask immediately, blows and beats repeatedly several times, it is outstanding to collect cell
Liquid, 1300rpm are centrifuged 5min, and cell is resuspended with fresh prawns complete medium, is inoculated into new Tissue Culture Flask.
(3) Tissue Culture Flask is placed in 28 DEG C, 3%CO2It is cultivated in incubator.
As shown in Fig. 8, when being passed on the digestive ferment HyQTase of cold PBS combination nonmammalian source, obtain compared with
Good passage effect.HyQTase stoste acts on cell monolayer 5 minutes, can be by about 90% or more cell from cell culture
It is digested in bottle, is inoculated into cell energy adherent growth after new bottle.
Claims (3)
1. a kind of cultural method of prawn embryonic cell, which is characterized in that the cultural method is the prawn embryo that will be separated
Cell suspension inoculation is into culture bottle or culture plate, in 28 DEG C, 3% CO2Stationary culture in incubator；Every 2 days one halfbodies of replacement
Long-pending prawn complete medium；
The prawn embryonic cell suspension, preparation method are as follows:
In the prawn embryo transfer to sterilized mortar that cleaning is saved, the extra culture medium of reject；It is thin with sterilized embryo
The curved stainless steel strainer of born of the same parents' collector squeezes prawn embryo to smash fertilization membrane to pieces, makes embryonic cell separate out；Then it takes new
Fresh prawn basal medium rinses stainless steel filtering net, collects all embryonic cells and cell mass, then cultivated with prawn basis
Base, sedimentation is washed 1-2 times, until clear, finally with prawn complete medium suspension embryonic cell and cell mass acquisition pair
Shrimp embryonic cell suspension；
The prawn basal medium the preparation method is as follows: dissolving 20.55 g L-15 culture mediums, 5 in 1 liter of ultrapure water
G NaCl, 2 g glucose, 1 g NaHCO3, 0.01 g taurine, 0.01 g proline, 1.0 × 105IU Benzylpenicillin sodium salt and
100mg streptomycin sulphate adjusts pH value to 7.2-7.4, with 0.22 μm of filtering with microporous membrane degerming, -20 DEG C of preservations；
The preparation method of the prawn complete medium is as follows: often going up and states in prawn basal medium, adds 8% tire ox blood
Clearly, 20 μ g basic fibroblast growth factors and 20 μ g epidermal growth factor, pH7.2-7.4；With 0.22 μm of miillpore filter
Preparation is completed in filtration sterilization；
The prawn embryo that the cleaning saves is that prawn embryo is collected with suction pipe into centrifuge tube, and antiseptic sea water washs 5-6
It is secondary, then by embryo transfer into 70 mesh stainless steel filtering nets, after eluriating for several times, flowing water continuous flushing 1-2 h, then with 10% Iodophor
Processing 5-10 minutes, then 20-30 seconds removal Iodophors are rinsed with 75% alcohol；Then successively with added with 2000 IU/mL penicillin
With antiseptic sea water and PBS the rinsing removal alcohol of streptomysin；It is finally temporarily stored into prawn basal medium.
2. the method as described in claim 1, which is characterized in that the antiseptic sea water is that natural sea-water is successively passed through 16-
After 20 layers of gauze and 0.22 μm of membrane filtration, made of high pressure steam sterilization.
3. the method as described in claim 1, which is characterized in that the preparation method of the PBS solution is as follows: weighing respectively
8.0 g NaCl, 0.2 g KCl, 3.0 g Na2HPO4·12H2The KH of O and 0.2 g2PO4It is dissolved in 1 L pure water, dissolves, with 3
M NaOH adjust pH value to 7.2 ~ 7.4,121 DEG C high pressure sterilization 20 minutes, saved backup for 4 DEG C after packing.
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| CN110551682B (en) * | 2018-06-01 | 2023-06-09 | 深圳华大生命科学研究院 | Kit and method for dissociation of animal embryos |
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| CN105754928A (en) | 2016-07-13 |
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